A strategy based on nucleotide specificity leads to a subfamily-selective and cell-active inhibitor of N⁶-methyladenosine demethylase FTO

Abstract

The AlkB family of nucleic acid demethylases are of intense biological and medical interest. The discovery of a highly selective FTO inhibitor should greatly facilitate the study of these enzymes.

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily‐Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

“…To the best of our knowledge, this study represents not only the first example of multiprotein DCC, but also an ew strategy for probingd ynamic chemicals ystems, which will hopefully furthero ur understanding of protein-directed self-assembly, and inspiren ew applications for DCC-based approaches. [53] )and ALKBH3( salmonsurface and salmons ticks;PDB ID 2IUW [54] ). The rightp anels show close-up views of interactions between 7 and active-site residues in FTO and ALKBH3.…”

“…Consistent with our detection strategy,t here was also am arked separation of individual meltingp eaks (DT m > 9 8C), whiche nabled any ligand-induced T m shift to be clearly detected without merging of peaks, as demonstrated by ligand binding experiments with 1 (a known generic inhibitor of AlkB subfamilies) [57] and FTO-selective inhibitor 2 (PubChem ID:1 26970771;F igure 3C and D). [53] By comparison, DSF analysis of am ixture of untagged ALKBH5, FTO, and ALKBH3 produced an overlapping melting profile that was difficult to deconvolute( Figure 3E). Thus, the combined use of thermalt ags with DSF enables the simultaneous analysiso fd ifferent fusion proteins in am ixture, even structurally similars ubfamily members,w hich is otherwise challenging to differentiate.…”

“…As our model dynamic reaction, we chose the reversible acyl hy- Figure 3. The thermal tags provide abasisfor differential analysis of several proteins simultaneously.T he DSF-based melting profiles of ALKBH5-KE6, FTO-KE12, and ALKBH3-KE18,upon being analyzed A) individuallyand B) as am ixture, revealed that the mixing of fusion proteins did not affect their individual melting behavior.Ligand binding experiments with C) generic inhibitor 2,4-pyridine dicarboxylic acid (1;100 mm) [57] and D) FTO-selectivei nhibitor 2 (100 mm) [53] demonstrated thecapability of the DSF detections trategy to monitorseveral protein-ligand interactions in as ingle DSF screen.E)DSF analysis of am ixture of unmodified ALKBH5, FTO, and ALKBH3 produced an overlapping melting profile to demonstrate the power of the thermal tag approachin discriminatings tructurally similars ubfamily members. drazoner eaction between scaffold ligand 3 and aldehydes ( Figure 4A).…”

“…Compound 2 was synthesized as previously reported. [53] The synthesis and characterization of selected compounds, 7, 8, 17, 21,a nd 22,a re described below.F or the synthesis of other compounds investigated herein, see the Supporting Information.…”

The role of chlorine atom on the binding between 2‐phenyl‐1H‐benzimidazole analogues and fat mass and obesity‐associated protein

“…FTO inhibitors have been reported, such as rhein, α‐ketoglutarate (αKG) analogues, meclofenamic acid, radicicol, and so on . Previous work in our group showed that 4‐chloro‐6‐(6'‐chloro‐7'‐hydroxy‐2',4',4,‐trimethyl‐chroman‐2'‐yl)benzene‐1,3‐diol (CHTB) and N‐(5‐chloro‐2,4‐dihydroxyphenyl)‐1‐phenylcyclobutanecarboxamide (N‐CDPCB) are inhibitors of FTO .…”